The voltage-sensitive sodium channel is responsible for the electrochemical action potential in nerve, neuroendocrine, skeletal muscle, and heart cells. The action potential is generated by a rapid increase in sodium permeability. Electrically excitable cells maintain a high intracellular K.sup.+ concentration and a low intracellular Na.sup.+ concentration (relative to the extracellular fluid) by energy-dependent cation pumping mediated by Na.sup.+ -K.sup.+ -ATPases. The resting membrane potential (inside negative) is maintained in excitable cells by surface membranes that are specifically permeable to K.sup.+. During generation of an axonal action potential, voltage-sensitive ion channels respond with large increases in permeabilities to specific ions on a time scale of milliseconds. Estimates imply physiological ion transport rates of &gt;10.sup.7 ions per second, consistent with the movement of Na.sup.+ through a fixed pore or channel. The depolarizing phase of the action potential results from an increase in sodium permeability, whereas repolarization and hyperpolarization following the action potential result from increases in potassium permeability. Sodium permeability changes during depolarization are biphasic with first a dramatic increase and then a decrease to the baseline level after .about. 1 ms. Thus, the sodium channel fulfills fundamentally different functions in cells than the potassium channel, and the electrochemical events mediated by the two channels are diametrically opposed but balanced.
It has been suggested that sodium channel "activation" controls the rate and voltage dependence of the sodium permeability increase during depolarization, while "inactivation" controls the rate and voltage dependence for the return to resting levels during a maintained depolarization. The sodium channel therefore appears to exist in three functionally distinct states or groups of states: resting, active, and inactivated. Both resting and inactivated states do not conduct ions, and channels that have been inactivated by prolonged depolarization are refractory unless the cell is electrically repolarized to allow them to return to the resting state. The rapid ion conductance by the sodium channel is remarkably selective, with potassium being only .about.8% as permeable as sodium, and rubidium and cesium even less permeant (1; see the appended Citations). These fundamental electrochemical properties of sodium channels are the foundation on which essentially all subsequent studies of sodium channel function have been based.
On theoretical grounds, the changes in sodium channel permeability during activation most likely result from conformational changes in one or more channel components. One or more membrane proteins may be induced to undergo a voltage-driven change in conformation to a new stable state in which the net charge, or the location of charge, within the membrane electrical field has been altered. A movement of membrane-bound charge can give rise to a measurable capacitive current detected as "gating currents" (2, 3) by using electrophysiological methods. Inactivation of sodium channels blocks gating currents.
Identification of sodium channel polypeptides was achieved by covalent in situ labeling of a neurotoxin receptor site 3 using a photoreactive azidonitrobenzoyl derivative of scorpion toxin (4). Two polypeptides of 260 kDa and 36 kDa were identified by SDS-PAGE that have subsequently been termed the .alpha.- and .beta.1-subunits. In contrast, the Triton X-100 solubilized sodium channel from rat brain reportedly has a Stokes radius of 80 .ANG., a sedimentation coefficient of 12 S, a partial specific volume of 0.82 cm.sup.3 /g (5), and a deduced molecular mass of about 601 kDa (i.e., for the protein-detergent complex). The purified rat brain sodium channel consists of three polypeptides: .alpha. of 260 kDa, .beta.1 of 36 kDa, and .beta.2 of 33 leda (6, 7, 8, 9). .beta.2 is covalently attached to .alpha. by disulfide bonds, and .beta., is associated noncovalently (8, 9) The subunits appear to be present in a 1:1:1 stoichiometry (7), and all three polypeptides are intrinsic membrane glycoproteins.
Nucleotide sequences encoding a sodium channel polypeptide were reported for an electric eel electroplax sodium channel (10). The deduced amino acid sequence revealed a protein with four internally homologous domains, each containing multiple potential .alpha.-helical transmembrane segments. The cDNAs encoding the electroplax sodium channel were used to isolate other cDNAs encoding three distinct, but highly homologous, rat brain sodium channels (types I, II, and III; 11, 12). The type II gene contains two adjacent exons that are alternatively spliced into mature mRNA in a developmentally regulated manner. The type II sodium channel is most prominent in embryonic and neonatal brain, whereas the type IIA channel is most prominent in the adult brain (13, 14). Each of these sodium channel .alpha.-subunits consists of four homologous domains. cDNAs encoding the alternatively spliced rat brain type IIA sodium channel .alpha.-subunit have been isolated (15, 16), and the type II/IIA cDNA was used as a probe at low stringency to isolate cDNAs encoding sodium channel .alpha.-subunits expressed in skeletal muscle and heart (17, 18, 19). The .mu.-sodium channel .alpha.-subunit is expressed primarily in adult skeletal muscle (19); the h1 sodium channel .alpha.-subunit is expressed primarily in heart and also in uninnervated or denervated skeletal muscle (17, 18). These sodium channels have the alpha helical transmembrane structural motifs similar to those in the .alpha.-subunits of the brain sodium channel.
The inactivation gate, which closes the sodium channel during prolonged depolarization, is formed by the intracellular protein segment connecting homologous domains III and IV. Antibodies against this segment prevent inactivation and therefore keep the channel open during prolonged depolarization (20, 21). Mutations which cut the protein between domains III and IV also slow inactivation (22). This segment serves to close the channel.